Prevention of peroxidase mediated inhibition of neutrophil motility and lymphocyte transformation by levamisole, OMPI, sodium aurothiomalate, indomethacin and tolmetin in vitro

The effects of sodium aurothiomalate, levamisole, its active metabolite OMPI and the anti-inflammatory agents indomethacin and tolmetin on neutrophil motility and post-phagocytic hexose monophosphate shunt activity, superoxide and H2O2 generation and myeloperoxidase (MPO) mediated iodination of Candida albicans were investigated in vitro. All five agents caused stimulation of neutrophil random motility and migration towards the leucoattractants f-met-met-phe and EAS. Only levamisole caused inhibition of H2O2 and superoxide production, which was associated with inhibition of HMS activity and not related to superoxide scavenging activity. All five agents caused inhibition of MPO mediated iodination of C. albicans. The relationship between inhibition of peroxidase mediated iodination and enhanced motility was further investigated using the horseradish peroxidase (HRP) H2O2/iodide system. Incubation of neutrophils with this system caused inhibition of neutrophil motility. However in the presence of the various drugs neutrophils were protected from inhibition of motility by the HRP/H2O2/iodide system. Further experiments showed that lymphocyte transformation to mitogens was also inhibited by the HRP/H2O2/iodide system. Incubation of lymphocytes with the various drugs prior to exposure to HRP/H2O2/iodide protected the lymphocyte mitogenic responsiveness.     

Increased leucoattractant binding and reversible inhibition of neutrophil motility mediated by the peroxidase/H2O2/halide system: effects of ascorbate, cysteine, dithiothreitol, levamisole and thiamine.

Human blood neutrophils manifested markedly decreased motility following exposure to the horseradish peroxidase (HRP)/H2O2/halide system in vitro. These cells were protected from this inhibitory effect (of the HRP/H2O2/halide system) by inclusion of concentrations in the reaction system of ascorbate, cysteinee, levamisole and thiamine which stimulate neutrophil migration and inhibit activity of the HRP/H2O2/halide system. The reversible nature of the oxidative inhibition of migration was demonstrated by exposing neutrophils to the HRP/H2O2/halide system for 15 min followed by washing to remove the components of the peroxidative system, and subsequent addition of ascorbate, cystein, levamisole, thiamine and the reducing agent, dithiothreitol. Neutrophils so treated completely recovered normal or increased motility induced by the leucoattractants endotoxin-activated serum or synthetic chemotactic tripeptide f-met-leu-phe. This reversible loss of migratory responsiveness following exposure of neutrophils to the HRP/H2O2/halide system was not associated with decreased cell viability or adherence. However, membrane oxidation was accompanied by increased uptake of radiolabelled f-met-leu-phe and degranulation. The increased leucoattractant uptake was decreased by ascorbate, levamisole and thiamine. These agents also prevented oxidation of the neutrophil membrane by the HRP/H2O2/halide system as measured indirectly by inhibition of iodination.     

In vitro stimulation of neutrophil motility by metoprolol and sotalol related to inhibition of both H2O2 production and peroxidase mediated iodination of the cell and leucoattractant

The effects of the beta-receptor blockading agents, metoprolol and sotalol on neutrophil random motility, chemotaxis, post-phagocytic glycolysis, superoxide production, hexose monophosphate shunt activity, myeloperoxidase (MPO) mediated protein iodination and hydrogen peroxide production were assessed in vitro. The concentration range investigated was 10(-8)--10(-2) M for each drug. Both agents caused significant stimulation of neutrophil motility at concentrations of more than 10(-4) M. Increased migration was not associated with increased glycolysis or significant cyclic nucleotide fluctuations, but was inversely related to inhibition of superoxide and hydrogen peroxide generation and MPO mediated iodination with both drugs. In a further series of experiments to determine the relationship between the drug induced inhibition of H2O2 production and MPO mediated protein iodination to stimulation of motility it was found that concentrations of sotalol and metoprolol that caused these effects prevented HRP/H2O2/I- induced inactivation of the leucoattractant and inhibition of neutrophil chemotactic responsiveness. Neither drug inhibited the activity of MPO per se nor the reduction of ferricytochrome c by superoxide generated by the xanthine: xanthine oxidase system in vitro. It is suggested that enhanced neutrophil motility is not related to beta-receptor blockade but rather to restricting the availability of hydrogen peroxide and reactive products of the MPO/H2O2/halide system.     

In vitro and in vivo stimulation of neutrophil migration and lymphocyte transformation by thiamine related to inhibition of the peroxidase/H2O2/halide system.

The effects of thiamine on neutrophil functions and mitogen-induced lymphocyte transformation were investigated in vitro and in vivo in adult volunteers following the injection of 50 mg thiamine intramuscularly. Thiamine caused stimulation of neutrophil motility in vitro and in vivo and increased lymphocyte transformation in vivo. Enhancement of these functions was related to inhibition of neutrophil post-phagocytic iodination of Candida albicans by the MPO/H2O2/halide system. The horseradish peroxidase/-H2O2/125 I-mediated iodination of bovine serum albumin was also inhibited by thiamine concentrations which caused increased neutrophil motility. It was found that preincubation of neutrophils and lymphocytes with the horseradish peroxidase/H2O2/halide system caused considerable inhibition of the migratory and proliferative responses respectively. Inclusion of thiamine at concentrations which were found to inhibit the peroxidase/-H2O2/halide system protected the neutrophil migratory and lymphocyte proliferative responses from inactivation by this system. It is suggested that thiamine may cause increased neutrophil migration and lymphocyte transformation by protecting these cells from toxic oxidative products generated by the peroxidase/H2O2/halide system.     

The in vitro effects of propranolol and atenolol on neutrophil motility and post-phagocytic metabolic activity.

Propranolol at concentrations of 1 x 10(-6) to 1 x 10(-4) M consistently increased neutrophil motility as measured in Boyden chambers. The effects were not due solely to stimulation of random migration and chemokinesis but also of directional motility. Propranolol, over a similar concentration range, caused inhibition of post-phagocytic cell metabolic activity (hexose monophosphate shunt, nitro-blue tetrazolium reduction and protein iodination) without any detectable effect on the ingestion rate of Candida albicans. Atenolol had no effect on any of these neutrophil functions. Both drugs were without effect on glycolysis and intracellular cyclic AMP levels. Propranolol however, at concentrations which stimulated cell motility, caused increased intracellular cyclic GMP levels. It is suggested that propranolol may stimulate neutrophil motility by promoting increased intracellular cyclic GMP levels or by decreasing neutrophil superoxide production.     

Stimulation of human neutrophil oxidative metabolism by nonopsonized Neisseria gonorrhoeae.

Nonopsonized gonococci possessing opacity-associated (Opa; previously PII) outer membrane proteins stimulate neutrophils to undergo a vigorous oxidative response when measured by luminol-dependent chemiluminescence (LDCL). In these studies, we characterized the mechanism of this stimulation. No gonococci that we tested induced measurable release of neutrophil superoxide anion (O2-) or hydrogen peroxide (H2O2) as measured by reduction of cytochrome c or the oxidation of scopoletin, respectively. Neutrophils pretreated with gonococci and then exposed to phorbol myristate acetate, the chemotactic peptide formylmethionylleucylphenylalanine, or opsonized zymosan released levels of neutrophil O2- and H2O2 comparable to controls, indicating that gonococci were not preventing or inhibiting neutrophil O2- or H2O2 release. To ascertain a possible explanation for these seemingly contradictory observations (i.e., induction of LDCL, but no release of O2- or H2O2), we further characterized the ability of Opa+ gonococci to stimulate LDCL. By using 1 mM azide and 4 U of horseradish peroxidase to monitor extracellular LDCL selectively and 2,000 U of catalase to monitor intracellular LDCL selectively, we determined that greater than 80% of total gonococcus-induced neutrophil LDCL occurred intracellularly. In addition, neutrophils stimulated with Opa+ gonococci showed a marked increase in O2 uptake and hexose monophosphate shunt activity. We conclude that Neisseria gonorrhoeae induces neutrophil oxidative metabolism without causing release of detectable amounts of reactive oxygen intermediates into the surrounding milieu. The gonococcus apparently directs oxidase assembly and activity to the phagolysosomal membrane. This could be a mechanism by which extracellular gonococci persist for extended periods in vivo in the presence of high concentrations of neutrophils.     

Preferential inhibition of primary granule release from bovine neutrophils by a Brucella abortus extract.

A low-molecular-weight Brucella abortus extract (a nucleotidelike material) inhibited zymosan-elicited neutrophil degranulation and trichloroacetic acid-precipitable protein iodination (a measure of myeloperoxidase and H2O2 release from neutrophilic leukocytes). Inhibition of neutrophil function was directly related to the concentration of the Brucella extract. The extract preferentially inhibited degranulation of primary (azurophilic or peroxidase positive) granules and had limited inhibition of secondary (specific or peroxidase negative) granule release but did not inhibit opsonized zymosan ingestion. Inhibition of protein iodination closely paralleled that of primary granule release but was unrelated to inhibition of secondary granule release. These results suggest that B. abortus has a component which is capable of inhibiting release of myeloperoxidase by dose-dependent preferential inhibition of primary granule release from bovine neutrophilic leukocytes.     

Peroxidase-mediated iodination by guinea pig peritoneal exudate eosinophils

Leukocyte iodination, which requires the granular enzyme peroxidase, hydrogen peroxide generated by the cell, and an appropriate oxidizable cofactor such as iodide, was studied in guinea pig peritoneal exudates. Eosinophils had an active resting iodination which was increased approximately 10-fold by the addition of serum and zymosan. In contrast, neutrophils had barely detectable resting iodination, but marked stimulation also occurred employing serum and zymosan or preopsonized zymosan. Under all conditions tested eosinophil iodination was significantly greater than neutrophil iodination (P< 0.001). The role of the peroxidase system in iodination was confirmed by inhibition in the presence of 10^–3 M azide, cyanide, or aminotriazole. Inhibition by exogenous catalase and facilitation by superoxide dismutase substantiated the role of hydrogen peroxide. Autoradiographic and cell separation studies confirmed that the majority of iodination was cell or particle associated.Dr. Pincus was a Research Associate of the Veterans Administration. Additional support was provided by a fellowship from the Dermatology Foundation and NIH grant 5 K08 AM 00538-02.     

Assessment of oral ascorbate in three children with chronic granulomatous disease and defective neutrophil motility over a 2-year period

Two brothers and their sister with chronic granulomatous disease, elevated levels of serum IgE and defective neutrophil motility were treated with a single oral daily dose of 1 g sodium ascorbate as a supplement to prophylactic trimethoprim--sulphamethoxazole therapy for 2 years. Laboratory tests of neutrophil functions were performed prior to ascorbate therapy and repeated at 1-monthly intervals for 6 months and at 6-monthly intervals thereafter. Introduction of ascorbate to the therapeutic regimen was accompanied by slight increases in neutrophil hexose monophosphate shunt activity and staphylocidal activity and good improvement of neutrophil motility in all three children. The improved staphylocidal activity was not due to ascorbate-mediated inhibition of neutrophil or serum catalase activities or to detectable increases in superoxide and H2O2 production or activity of the MPO/H2O2/halide system. Both male children have remained free from obvious infection since ascorbate was added to their therapeutic regimen; their sister has experienced one urinary tract infection during a period when treatment with prophylactic co-trimoxazole and ascorbate was inadvertently stopped. All three children have gained weight.     

Clofazimine-mediated regulation of human polymorphonuclear leukocyte migration by pro-oxidative inactivation of both leukoattractants and cellular migratory responsiveness

The effects of clofazimine (0.15-20 micrograms/ml) on the spontaneous and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated migration, membrane-associated oxidative metabolism, degranulation and production of prostaglandin (PG) E2 by human polymorphonuclear in vitro have been investigated. Clofazimine at concentrations of 0.3 microgram/ml and greater significantly increased both the spontaneous and FMLP-stimulated chemiluminescence (CL), hexose monophosphate shunt (HMS) activity, myeloperoxidase-mediated protein iodination, auto-iodination, degranulation and PGE2 production by PMNL. At the same concentrations clofazimine inhibited both random and leukoattractant-induced migration of PMNL. Inhibition of PMNL migration by clofazimine was due to both a cell-directed auto-oxidative mechanism and by functional inactivation of FMLP. Clofazimine mediated inhibition of PMNL migration was prevented by the anti-oxidants cysteine and dapsone but not by the potent inhibitors of PG synthetase indomethacin and piroxicam. Anti-oxidants also protected FMLP from functional inactivation by clofazimine-exposed PMNL. Clofazimine increased both the spontaneous and FMLP-stimulated production of PGE2 by PMNL from four children with chronic granulomatous disease (CGD). Clofazimine is not an oxidising agent nor did it stimulate membrane-associated oxidative metabolism in CGD or NaF-pulsed normal PMNL. These data show that clofazimine-mediated inhibition of PMNL migration is dependent on intact cellular membrane-associated oxidative metabolism. Clofazimine is therefore a pro-oxidative anti-inflammatory agent.     

The in vitro evaluation of certain neutrophil and lymphocyte functions following the ingestion of 150 mg oral dose of levamisole: assessment of the extent and duration of stimulation of neutrophil chemotaxis, protein iodination and lymphocyte transformation.

Certain functions of human blood neutrophils and lymphocytes were investigated at varying time intervals after the ingestion of a single 150 mg dose of levamisole. The functions tested were neutrophil chemotaxis and post-phagocytic metabolic activity and mitogen-induced DNA and protein synthesis of lymphocytes. It was found that levamisole causes a stimulation of neutrophils motility (cell- and serum-associated) and post-phagocytic hexose monophosphate shunt activity and protein iodination. Increased lymphocyte DNA synthesis, but not protein synthesis, to the mitogen phytohaemagglutinin was observed. The stimulation which was detected almost immediately of these neutrophil and lymphocyte functions was still evident 24 hr later but not at 48 hr, indicating that a single oral dose of levamisole can cause the alteration (stimulation) of leucocyte functions which persists until 24--48 hr after intake of the drug.     

Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa

Lipid peroxidation occurs in human sperm cells with damage to the cell plasma membrane, leading to loss of cytosolic components and hence to cell 'death'. The peroxidation may be induced at high rates in the presence of Fe2+ and ascorbate. It occurs at slower rates under physiological conditions as spontaneous lipid peroxidation, which has the following characteristics. The rate is constant over the time required for complete loss of motility in the cells of the sperm sample; one can thus use the time to complete loss of motility (TLM) as a ready measure of the rate. Loss of motility occurs at a characteristic extent of lipid peroxidation, assayed in terms of production of the peroxidative breakdown product, malonaldehyde (MA), that is independent of peroxidation rate. For human sperm, this extent corresponds to 0.1 nmol MA/10(8) cells. Human spermatozoa possess the anti-lipoperoxidative defence enzymes, superoxide dismutase (SOD) and glutathione peroxidase plus glutathione reductase (GPX/GRD). The SOD activity is highly variable between human sperm samples while the activities of GPX and GRD are rather more constant. The rates of production of superoxide anion, O2-, and hydrogen peroxide, H2O2, from human spermatozoa are variable, but their sum calculated in O2- equivalents as O2- + 2H2O2 is quite constant. The variability arises from the variability in SOD activity: all H2O2 produced is from O2- due to the action of SOD. The essential role of SOD as defence enzyme is inferred from the observation that TLM of a given sperm sample is directly proportional to the SOD activity of that sample. The essential role of GPX/GRD is inferred from the observation that inhibition of GPX, either with mercaptosuccinate or with complete oxidation of intracellular reduced glutathione, results in a 20-fold increase in peroxidation rate. The capacity of the GPX/GRD system appears to be limited by the glucose-6-phosphate dehydrogenase-catalysed rate of production of NADPH, the required reductive substrate for GRD. Human spermatozoa appear to have enough anti-lipoperoxidative defensive capacity for lifetimes long enough for fertilization but still short enough for ready removal from the female reproductive tract in good time. Too low a defence capacity could lead to male infertility.      

The comparative study of reactive oxygen species generated by polymorphonuclear leukocytes as alpha 1-proteinase inhibitor inactivators-possible application for antioxidant prevention of emphysema

The oxidative inactivation of alpha 1-proteinase (alpha 1AP) inhibitor is a one of mechanisms that may lead to the pulmonary emphysema. This process is caused by oxidants derived from atmosphere and released from lung phagocytes. These cells produce various oxidants hydrogen peroxide (H2O2), hypochlorous acid (HClO), hydroxyl (OH.) and superoxide (O2-) radicals after inflammatory stimulation. In this study I have investigated the effects of H2O2 (1.5 x 10(-5) to 1.5 x 10(-2) M) alone or with addition of FeCl2 (50 microM) in order to generate OH., chloramine-T (1.5 x 10(-5) to 1.5 x 10(-3) M) which generates HClO, glucose 10 mg/ml-glucose oxidase (12.5 to 80 mU/ml)-H2O2 generating system, xanthine 0.2 mM-xanthine oxidase (12.5 to 80 mU/ml)-O2-2 generating system on the elastase inhibitory activity of alpha 1AP in vitro. H2O2 was weak in alpha 1AP inactivation--only concentration of H2O2 1.5 x 10(-2) caused severe loss of its activity to 23 +/- 8% inhibition of elastase. Addition of FeCl2 to H2O2 and following OH. generation did not enhance its alpha 1AP inactivation. O2-2 generating system inhibited moderately alpha 1AP. The % inhibition of elastase at concentration of xanthine oxidase 80 mU/ml was 65 +/- 7. HClO was most effective as an alpha 1AP inactivator. All used chloramine-T concentrations completely suppressed alpha 1AP activity. The obtained results and in vivo consumption of H2O2 by polymorphonuclear leukocyte myeloperoxidase for HClO production suggest that scavenging of these reactive oxygen species may be useful in prevention of emphysema.     

Oxidative inactivation of Actinobacillus actinomycetemcomitans leukotoxin by the neutrophil myeloperoxidase system.

The leukotoxin of Actinobacillus actinomycetemcomitans has been implicated in the pathogenesis of inflammatory periodontal disease. We examined a potential mechanism for detoxification of this microbial product by the neutrophil myeloperoxidase system. Exposure to myeloperoxidase, H2O2, and a halide resulted in marked inactivation of leukotoxin, an effect which required each component of the myeloperoxidase system. Toxin inactivation was blocked by agents which inhibit heme enzymes (azide, cyanide) or degrade H2O2 (catalase). Reagent H2O2 could be replaced by the peroxide-generating enzyme system glucose oxidase plus glucose. The latter system, in fact, was more potent than reagent H2O2 in terms of the capacity to inactivate high concentrations of toxin. Toxin inactivation was complete within 1 to 2 min at 37 degrees C. These observations suggest a possible role for oxidative inactivation of leukotoxin by secretory products of neutrophils.     

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.     

Pleiotropic effects of the Bcg gene: III. Respiratory burst in Bcg-congenic macrophages.

Macrophage respiratory burst, as assessed by H2O2 and O2- production, and HMS and chemiluminescence activity was investigated in a variety of conditions in macrophages from Bcg-congenic mice. Measurement of HMS and chemiluminescence in splenic macrophages challenged in vitro with BCG showed that the Bcgr cells were more stimulated by the challenge than their Bcgs counterparts. H2O2 production was measured in Bcgr and Bcgs splenic macrophages. PMA-triggering and LK-triggering were shown to stimulate a similar degree of H2O2 production Bcgr and Bcgs macrophages. In contrast, in vitro phagocytosis of BCG was shown to trigger superior production of H2O2 and O2- in the Bcgr splenic macrophages as compared to their Bcgs congenics. Finally, following the in vivo infection with BCG Montreal, Bcgr splenic macrophages were superior producers of H2O2 (both spontaneous and PMA-triggered) in the early phase of infection.     

Generation of hydrogen peroxide by Candida albicans and influence on murine polymorphonuclear leukocyte activity.

Iodide fixation by murine polymorphonuclear leukocytes (PMN) incubated with viable Candida albicans blastoconidia increases directly with yeast cell concentration up to about 3 x 10(6) cells per ml, but above this concentration bound activity declines dramatically. To understand the basis for this decline, we examined the oxidative metabolism of fungi and stimulated PMN and found some remarkable similarities between these cell types. Both produced 14CO2 when incubated with [1-14C]glucose, both reduced cytochrome c, and both fixed radiolabeled iodide, although the fungi required exogenous lactoperoxidase. In dose-response experiments, iodination by fungi with lactoperoxidase was identical to that with PMN, i.e., the maximum bound activity occurred in cultures with 10(6) to 3 x 10(6) blastoconidia per ml. Iodination by fungi with lactoperoxidase was reduced when blastoconidia were incubated at 25 degrees C or in the presence of catalase and the metabolic inhibitors rotenone, antimycin A, and 2-deoxyglucose. Results from assays for oxidation of scopoletin and o-dianisidine showed that 10(6) blastoconidia in 1.0 ml of medium released 0.5 to 0.7 nmol of H2O2 after 1 h, but 3 X 10(6) and 10(7) cells released significantly less H2O2. These results suggest that iodide fixation by PMN and low numbers of fungal cells may reflect a cooperative effort, with fungi generating some H2O2 that reacts with the myeloperoxidase released from the PMN. With high concentrations of blastoconidia, H2O2 activity appeared to be specifically inhibited, possibly to protect fungal cells from damage.     

The mechanism of action of lymphokines. VIII. Lymphokine-enhanced spontaneous hydrogen peroxide production by macrophages.

Oil-elicited guinea-pig peritoneal macrophages (MPs) cultured for 2-3 days in medium containing supernatant of concanavalin A-activated lymphocytes (lymphokine, LK) generated large amounts of hydrogen peroxide (H2O2), as detected by the horseradish peroxidase (HRP)-dependent oxidation of phenol red, in the absence of further stimulation. H2O2 production increased with the duration of exposure to LK and was evident at high dilutions of supernatant (1/64). Parallel cultures of MPs in medium or a supernatant of non-activated lymphocytes also increased their H2O2 production during culture but levels at all time intervals were significantly lower than those measured in LK treated cultures. The marked increase in H2O2 production was associated with only a moderate increment in superoxide (O-2) liberation and this was not specific for LK treated cells. Detection of LK-dependent H2O2 production was dependent on ongoing pinocytosis during the assay. This and other arguments suggest that the HRP-phenol red assay, as applied here, detects H2O2 generation occurring at the level of intracellular vesicles and it is concluded that LK elicits H2O2 production that is limited to the intracellular compartment. H2O2 is, apparently, derived by non-enzymatic dismutation of O-2 taking place within the cell; LK treatment of MPs also resulted in a significant reduction in catalase activity.     

Dermatitis herpetiformis: Effects of sulfones and sulfonamides on neutrophil myeloperoxidase-mediated iodination and cytotoxicity

The effects of sulfones and sulfonamides on neutrophil myeloperoxidase-mediated iodination and cytotoxicity were studied usingin vitro assays to measure these parameters. Leukocyte iodination was documented using a quantitative assay to measure the iodination of protein by human neutrophils undergoing phagocytosis. Cytotoxicity for the tumor cell line LSTRA by human neutrophils activated by exposure to phorbol myristate acetate was measured by a⁵¹Cr release assay. Dapsone, diasone, and sulfapyridine, at concentrations comparable to serum levels obtained by therapeutic doses of drug, effectively inhibited iodination and cytotoxicity mediated by human neutrophils. Other sulfonamides showed little inhibition of either iodination or cytotoxicity. The amount of inhibition was comparable to that seen with the inhibitors azide or cyanide and occurred in a dose dependent manner with all three drugs. A cell-free cytotoxic system using myeloperoxidase, iodide, a H₂O₂ generating system, and target cells also showed inhibition by dapsone, diasone and sulfapyridine in a similar fashion. The active drugs inhibited both the intra- and the extracellular myeloperoxidase-H₂O₂-halide cytotoxic systems. Serial iodination studies of four dermatitis herpetiformis patients, evaluated while taking dapsone or sulfapyridine, showed inhibition of iodination by either drug. Levels of IgA immune complexes, as measured by the Raji cell radioimmune assay adapted for IgA, did not change when medication was withheld. These studies demonstrate that dapsone, diasone, and sulfapyridine inhibit both neutrophil iodination and cytotoxicity for tumor cells, while other sulfonamides have no effect. This confirms previous studies showing inhibition by myeloperoxidase mediated iodination by dapsone. Furthermore, the effect on neutrophils is quickly reversible;in vivo administered drug has no effect onin vitro function. The active drugs inhibit both intra- and extracellular cytotoxic systems. This may represent an important mechanism by which these drugs produce their therapeutic effects when used to treat inflammatory skin diseases.     

Some enzymatic characteristics of eosinophil peroxidase from patients with eosinophilia and from healthy donors.

Some enzymatic characteristics of human eosinophil peroxidase were compared with those of human myeloperoxidase. Both enzymes catalyzed the oxidation of iodide by hydrogen peroxide. This assay proved to be very sensitive; the activity of 100 eosinophils/ml could be measured. The position of the pH optimum of this reaction was linearly dependent on the logarithm of the iodide/H2O2 ratio. At the same substrate ratio, this optimum was about 1 pH unit higher for eosinophil peroxidase than for myeloperoxidase. This difference may be related to the action of myeloperoxidase inside an acidified phagolysosome as opposed to the extracellular action of eosinophil peroxidase on the surface of certain parasites. Under defined conditions (KI, 1.4 mM; H2O2, 0.18 mM; cetyltrimethylammonium bromide, 0.008% [wt/vol]; pH 6), the activity of eosinophil peroxidase could be measured in a mixed granulocyte suspension independently of myeloperoxidase. Eosinophils from patients with eosinophilia were found to contain as much peroxidase activity as did eosinophils from healthy donors. No enzymatic differences in eosinophil peroxidase were found between the two types of donors.